The toxicity of ionizing radiation (IR) in mammals is largely due to damage to the most radiosensitive tissues, the gastrointestinal (GI) tract and the hematopoietic (HP) system. A number of approaches and compounds have been developed to protect or restore HP, including immune, function following IR exposure. In contrast, GI radiation syndrome remains poorly treatable resulting in significant mortality and reduced quality of life for survivors. In preliminary studies it was demonstrated that the bacterial flagellin protein, acting as an NF-?B activating agonist of Toll-like receptor 5 (TLR5), is a powerful radioprotectant and radiomitigator capable of improving survival when given either before or after lethal IR, respectively. Notably, the anti-radiation effects of flagellin are associated with protection of the GI tract as well as the HP system. A pharmacologically optimized TLR5 agonist with reduced immunogenicity (CBLB502) was generated by deleting portions of flagellin that are non-essential for TLR5 activation. CBLB502 is effective and non-toxic in mice and non-human primates and is being developed into a stable, self-administrable radiation antidote projected for biodefense applications. This proposal is focused on deciphering the molecular and cellular mechanisms of action of CBLB502. Aim 1 will involve identification of cellular mediators of CBLB502's effect on protection and healing of small intestine in irradiated mice with particular attention on the role of bone marrow-derived cells. Aim 2 will be devoted to analysis of molecular signaling pathways induced by CBLB502 that are critical for its radioprotection and radiomitigation properties. A combination of hypothesis-driven and unbiased functional screening approaches will be used to determine CBLB502 molecular targets. Finally, Aim 3 will concentrate on assessing the effects of CBLB502 on the short- and long-term pathological consequences of IR in other radiosensitive tissues, including liver, kidney and lung. The frequency and severity of fibrosis and cancer development will be assessed in a mouse model. Completion of this program will provide critical information about an emerging class of radiation antidotes that will allow improved understanding of their potential as human therapeutics targeting acute radiation syndrome. The potential scenarios for nuclear accidents and terrorist attacks in today's world imply mass radiation injury casualties affected by systemic exposure to radioactive materials. The toxicity of total body irradiation (TBI) is associated with induction of acute radiation syndrome (ARS) primarily involving damage to the highly radiosensitive gastrointestinal (GI) tract and hematopoietic (HP) system. While a number of approaches and compounds have been developed to protect or restore HP function following TBI, GI radiation syndrome remains poorly treatable, resulting in significant mortality and reduced quality of life for survivors. An ideal radiation countermeasure would protect both the GI and HP systems and would be capable of both radioprotection (prophylaxis) and mitigation of radiation injury. Moreover, biodefense applications require a drug that is easily self-administered, has a long shelf life and does not require special handling or storage conditions. We have recently discovered a new class of agents that satisfy all of the above requirements, most notably being effective against GI radiation syndrome as well as HP syndrome. Our lead compound, named CBLB502, is a powerful radioprotectant and mitigator of radiation injury suitable for intramuscular injections. It is a polypeptide derived from bacterial protein flagellin that acts as an agonist of Toll-like receptor 5 (TLR5), which is expressed in radiosensitive cells of the intestine and bone marrow progenitors and, upon binding with the ligand, activates several pro-survival pathways, mediated by NF-?B signaling. Here we propose a program to comprehensively characterize the molecular and cellular mechanisms underlying the anti-radiation activity of CBLB502, particularly focusing on GI radiation syndrome. We will also investigate the effect of CBLB502 on the pathological consequences of IR in other tissues, including radiation carcinogenicity. The proposed experiments will provide essential mechanistic insight into the previously observed radio-protective and -mitigative effects of CBLB502.